Inactivation of genes by CpG island methylation has emerged as a major mechanism in the etiology of adenocarcinoma of the lung in both smokers and never smokers. Our studies during the initial funding period for this grant revealed a higher prevalence for silencing of the O6-methylguanine-DNA methyltransferase gene in never smokers compared to smokers. Recent studies have demonstrated that the cytosine-DNA methyltransferases (DNMT) play a key role not only in de novo methylation of promoter regions, but also in the recruitment of deacetylated histones to establish heterochromatin over the gene promoter region that denies access to regulatory proteins needed for transcription. While our own studies have demonstrated an important role for DNMT1 in lung cancer development, the contribution of DNMT3a and 3b has not been clearly substantiated. Modification of cytosines and the histone code through methylation are both important factors in gene silencing;however, it is not clear which process initiates the cascade of events that culminate in transcriptional repression. The factors that both target genes for silencing and the trigger that allows either CpG methylation and/or histone modification to ensue are unknown. Our studies described within this application have led us to hypothesize that a decrease in gene transcription and the establishment of a "methylation mark" within the gene promoter leads to gene silencing. This renewal will focus on the elucidation of gene targets and pathways involved in adenocarcinoma and define mechanisms involved in targeting, initiation, and propagation of gene-specific silencing through aberrant promoter hypermethylation. These goals will be accomplished through three specific aims. Specific Aim 1 will identify the methylation profile in adenocarcinomas from smokers and never smokers for novel genes discovered through a microarray approach and known genes involved in controlling cell cycle, apoptosis, growth control/differentiation and invasion. Specific Aim 2 will identify the role that the de novo cytosine-DNA methyltransferases 3a and 3b play in neoplastic transformation and gene-specific aberrant promoter hypermethylation. Human telomerase immortalized bronchial epithelial cells containing exogenous DNMT3a or 3b under the control of an inducible promoter will be exposed chronically to tobacco carcinogens. Finally, Specific Aim 3 will identify factors involved in establishing repressed chromatin around promoter regions for the p16 and death associated protein kinase genes. Transient transfection of minimized promoters into cell lines with an endogenously active or silenced gene will be used to elucidate the sequence of changes involved in repression of promoter activity. REVISED: July 7, 2005